Many neuroendocrine peptides and proteins are produced from precursors by a series of endopeptidase and carboxypeptidase reactions. Carboxypeptidase E (CPE) was initially thought to be the only carboxypeptidase required for the generation of most neuroendocrine peptides. Recently we found that fat/fat mice lack CPE activity due to a point mutation in the coding region of the gene. These mice are overweight, and males are hyperglycemic. Although peptide processing by the carboxypeptidase step is reduced in these mice, it is not eliminated despite the complete absence of functional CPE. In a search for novel enzymes with CPE-like properties, we recently discovered carboxypeptidase D (CPD). Based on our studies over the past three years, we hypothesize that CPD contributes to the processing of neuroendocrine peptides as well as proteins that transit the secretory pathway. In the present application, we will continue to test this hypothesis, as well as to explore the possibility that CPD performs additional functions in the cell. In Specific Aim 1, we will compare the enzymatic properties of the three carboxypeptidase-like domains of CPD. Since the third carboxypeptidase- like domain of CPD does not appear to be enzymatically active, we will explore other potential functions for this domain. In Aim 2, we will examine the molecular basis for the forms of CPD found in the cell, and in various tissues. Previously, we have found evidence for multiple forms of CPD. In Aim 3, we will address the physiological function(s) of CPD by overexpressing this enzyme in cell lines that are deficient in CPE, and then testing whether elevated CPD can further compensate for the deficiency of CPE. In addition, we will use various strategies to reduce levels of CPD (inhibitors, antisense RNA) in cultured cells and then examine the effect on peptide/protein processing and sorting. Together, this concerted approach will provide important information regarding the role of CPD in the production of neuroendocrine peptides.